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HAYATI Journal of Biosciences 23 (2016) 79e84

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Original research article

Construction of RNA Interference Vector to Silence AluminumTolerance Gene Candidate in Rice cv Hawara Bunar

Windarti Wahyuningtyas,1 Miftahudin Miftahudin,2* Utut Widyastuti,2

Aris Tjahjoleksono2

1 Plant Biology Graduate Program, Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Bogor, Indonesia.2 Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Bogor, Indonesia.

a r t i c l e i n f o

Article history:Received 27 January 2015Accepted 3 December 2015Available online 19 July 2016

KEYWORDS:30UTR_B11,Al tolerant,rice,RNAi vector

* Corresponding author.E-mail address: miftahudin@ipb.ac.id (M. MiftahuPeer review under responsibility of Institut Perta

http://dx.doi.org/10.1016/j.hjb.2016.06.0031978-3019/Copyright © 2016 Institut Pertanian Bo(http://creativecommons.org/licenses/by-nc-nd/4.0

a b s t r a c t

One of the aluminum (Al) tolerance gene candidates, namely B11 gene, has been successfully isolatedfrom Al-tolerant rice cv Hawara Bunar. However, the role of the gene in Al tolerance in rice has not beenknown. RNA interference (RNAi) technique is an effective tool to examine the biological function of thetarget gene in plant. The objective of the research was to construct RNAi recombinant vector carryinguntranslated region of the B11 gene. RNAi recombinant vector carrying 195 bp sized 30UTR_B11 fragmentas a double-stranded RNA (dsRNA) trigger has been successfully constructed using GATEWAY™ cloningtechnology, pENTR™/D-TOPO® as a shuttle vector, and pANDA vector as a destination vector. RNAiconstruct was successfully introduced into Agrobacterium tumefaciens AgL0, and has been infected to ricecv Hawara Bunar. Analysis of putative transgenic rice showed eight of 20 plants were transgenic carryingthe B11-RNAi construct.Copyright © 2016 Institut Pertanian Bogor. Production and hosting by Elsevier B.V. This is an open access

article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Rice (Oryza sativa L.) is one of the important food crops inIndonesia. Rice consumption continues to increase as the Indone-sian population increases. There are many constraints to meet thisdemand, which one of them is the continuous decreasing of fertilerice field areas due to its conversion into industrial and residentialareas. Maintaining and increasing rice production through riceextensification tomarginal land such as acid soil are the continuousefforts that should be taken to meet the rice demand in Indonesia.

Rice farming in acid soils is experiencing many obstacles, one ofthem is high solubility of aluminum (Al) that can be toxic for riceroot that considerably inhibits root growth and damages root tips(Kochian 1995). As a result, the plant roots will be inhibited innutrient and water uptake that consequently will reduce plantgrowth and production (Delhaize & Ryan 1995).

Development of tolerant rice varieties to Al toxicity can beachieved through both conventional breeding and genetic engi-neering approaches. Genetic engineering technology gives possi-bility to manipulate the plants to produce new varieties with

din).nian Bogor.

gor. Production and hosting by/).

superior traits, including rice tolerant to Al stress. Identification offunctional Al tolerance gene candidate is a preliminary step toachieve the goal. The function of a gene in plants can be studiedthrough two approaches. First, one can increase the expression ofthe gene by constructing over-expression vector. Second, to stop ordecrease the expression of the gene such as by constructing RNAinterference (RNAi) vector. Once the gene function is characterized,it can be used either for marker assisted selection or for trans-formation of rice into Al-tolerant transgenic plant.

The RNAi technique is an effective way to test the biologicalfunction of mRNA target in plants. Recent developments regardingthe RNAi vector, using constitutive promoter and Gateway™ clon-ing technique, makes it easy to construct the RNAi vector that has adouble-stranded RNA (dsRNA) trigger sequences and also makes iteasier to analyze the function of the target gene. RNAi causesdegradation of mRNA that genes become dysfunctional.

Effort to isolate Al tolerance gene from rice has been initiatedthrough revealing the syntenic relationship between Al tolerancelocus region in chromosome 4RL in rye and a short region in shortarm of rice chromosome 3 (Miftahudin et al. 2005). Throughscreening several markers in that rice region, Roslim (2011) hassuccessfully isolated an Al tolerance gene candidate from anIndonesian local rice cv Hawara Bunar. The gene was then clonedinto pGWB5 expression vector under 35S promoter and

Elsevier B.V. This is an open access article under the CC BY-NC-ND license

Table 2. Primer combinations used to check insert orientation in recombinantpANDA vector

No. Primer combination

Forward Reverse

1 GUS_cek-F 30UTR_B11-R2 GUS_cek-F 30UTR_B11-F3 GUS_cek-R 30UTR_B11-F4 GUS_cek-R 30UTR_B11-R

W. Wahyuningtyas, et al80

temporarily named as B11 gene. This article reports our effort tocharacterize the Al tolerance gene candidate function throughconstruction of RNAi vector.

2. Materials and Methods

2.1. MaterialsB11 clone in pGWB5, pENTR™/D-TOPO® cloning kit (Invi-

trogen™, USA) and pANDA vector (Prof. Ko Shimamoto collection,NAIST, Japan) were used for cloning the 30UTR and construction ofRNAi recombinant vector. The Escherichia coli DH5a and Agro-bacterium tumefaciens AgL0 were used as host for recombinantpENTR vector and recombinant pANDA vector, respectively. Rice cvHawara Bunar seeds was used to develop transgenic plants.

2.2. Isolation of 3′UTR fragmentThe 3' untranslated region of B11 gene (3'UTR_B11) fragment

was polymerase chain reaction (PCR) amplified from B11 gene clone(Roslim 2011) using primers 30UTR_B11-F and 30UTR_B11-R(Table 1). To facilitate the insertion of the 3'UTR_B11 fragment inthe cloning site of pENTR™/D-TOPO® vector, it is necessary to add aspecific sequence CACC at the 50 end of forward primer 30UTR_B11-F. The 20 mL PCR reaction consisted of 100 ng vector DNA template,1X PCR buffer, 0.2 mM dNTPs, 0.4 mM each primer and 1 U Taqpolymerase. The PCR program was 94�C for 5 minutes pre-dena-turation followed by 35 cycles of 94�C for 60 seconds denaturation,56�C for 30 seconds annealing, 72�C for 30 seconds extension. ThePCR was ended by 72�C for 10 minutes final extension. The PCRproducts were electrophoresed on 1% agarose gel (LE GQ TopVision, Fermentas, Canada) in 1X Tris/borate/EDTA (TBE) buffer at75 volt for 1 hour. The gel was then stained with ethidium bromide.Gel was observed and documented using WiseDoc Gel Documen-tation System (Daihan Scientific, South Korea). The 30UTR_B11fragment was eluted and purified from agarose gel following theprocedure of Geneaid gel purification kit (Geneaid, Taiwan).

2.3. Gateway cloning of 3′UTR_B11The cloning procedure of 30UTR_B11 fragment was carried out

based on Gateway Cloning System protocol (Invitrogen™, USA).First, the 30UTR_B11 fragment was inserted into a shuttle vectorpENTR™/D-TOPO® following the protocol provided in pENTR™/D-TOPO® cloning kit (Invitrogen™, USA). The ligated 30UTR_B11-pENTR™/D-TOPO® vector was then transformed into E. coli DH5ausing heat shock method (Sambrook et al. 1989). Transformantbacteria were grown in selection media Luria agar (LA) containing50 ppm kanamycin for overnight at 37�C. Several growth coloniesof bacteria were randomly selected for colony PCR following theprocedure performed by Suharsono et al. (2008) using a primer pairof 3'UTR_B11-F and 30UTR_B11-R. The vectors from positive col-onies carrying recombinant pENTR™/D-TOPO® e 30UTR_B11 vectorwere isolated using the Wizard Minicolumn Plasmid Extraction kit(Promega, USA) and then sequenced.

Second, the 30UTR_B11 fragment was then cloned from the re-combinant pENTR™/D-TOPO®-30UTR_B11 into pANDA vector using

Table 1. Primers used in the research on RNAi vector construction

Primers Sequences

30UTR_B11-F 50-CACCCTG TTTCTCCCCAAGTTCAG-30

30UTR_B11-R 50-GTCACGAGTGGCATTTGAG-30

GUS-F 50-CATGAAGATGCGGACTTACG-30

GUS-R 50-ATCCACGCCGTATTCGG-30

GUS_cek-F 50-CCGAATACGGCGTGGAT-30

GUS_cek-R 50-CGTAAGTCCGCATCTTCATG-30

GUS_cek-F and R are the reversed primer sequence of GUS-F and R.

the Gateway® LR Clonase ®II enzyme mix (Invitrogen™, USA) toobtain a recombinant expression vector (pANDA recombinant).Introduction of recombinant pANDA vector into competent E. coliDH5a and PCR colony followed the same procedure as describedpreviously, except the transformants were grown on LA selectionmedia containing 50 ppm of kanamycin and 50 ppm of hygromycin.Colony PCR test was performed using several primer combinations(Table 2).

2.4. Introduction of RNAi construct into A. tumefaciens AgL0Introduction of recombinant pANDA vector (RNAi construct)

into A. tumefaciens AgL0 was done by freeze-thaw methodfollowing the procedure performed by Xu and Li (2008). Thetransformants then were selected on LA medium containing50 ppm of kanamycin, 50 ppm of hygromycin, and 25 ppm ofrifampicin. Colony PCR was done using the same procedure asdescribed previously.

2.5. Infection of rice embryo through in-plantatransformation system

Introduction of RNAi construct into rice seeds cv Hawara Bunarwas carried out using in-planta transformation techniquefollowing the procedure of Lin et al. (2009). The bacteria wasinfected to the embryo or the plumule apical meristem usingsterile needles deeped in A. tumefaciens AgL0 culture. The infectedseeds were then planted in pots containing soil media and grownin a green house. The putative transgenic tillers and plantswere selected using PCR technique with GUS-F and GUS-R primersand HPT-F and HPT-R primers as well using the same PCRprocedure as mentioned previously. The positive tillers or plantswere then maintained to produce T1 seeds.

3. Results

3.1. Isolation of the 3′UTR fragment of B11 geneThe 30UTR_B11 fragment was successfully amplified from the 30

untranslated region of the B11 gene clone that previously has beencloned in pGWB5 (Figure 1A). After being visualized using agarosegel, the fragment was then purified and re-checked in agarose gel(Figure 1B). The fragment length of the unpurified and purified PCRproducts was the same as the expected length of the 30UTR B11gene sequence, suggesting that the PCR products were the 30UTR ofB11 gene. The fragment was then inserted in the pENTR™/D-TOPO®

shuttle vector and maintained in E. coli DH5a.

Function

Amplification of 30UTR fragment and check on insert orientationAmplification of 30UTR fragment and check on insert orientationCheck on GUS linker and transgenic plantCheck on GUS linker and transgenic plantCheck on insert orientationCheck on insert orientation

M C UTR UTR

1111

500 bp

200 bp

M C UTR UTR

500 bp

200 bp

A B

Figure 1. Electrophoregram of polymerase chain reaction (PCR) fragment amplified from 30UTR_B11 gene (A) and purified PCR fragment (B). The fragments were electrophoresed in1% agarose gel in 1X Tris/borate/EDTA (TBE) buffer at 75 volt for 45 minutes. C ¼ negative control; M ¼ 100 bp marker; UTR ¼ 30UTR_B11.

RNAi silencing of aluminum tolerance gene in rice 81

3.2. Recombinant pENTR-3′UTR_B11The recombinant vector of pENTR-30UTR_B11 was transformed

into E. coli DH5a and subsequently grown on LA medium contain-ing antibiotic. The transformation was successful as indicated bythe colonies grew on antibiotic selection medium. The growthcolonies were then verified using colony PCR and insert sequencingof the isolated recombinant vector. PCR verification of several col-onies using primers 30UTR_B11-F and 30UTR_B11-R showed thefragments with the same length as the original size (Figure 2).Sequence analysis of the insert showed the same sequence as theoriginal 30UTR sequence of B11 gene (Figure 3), which confirmedthat the 30UTR was successfully isolated from B11 gene and clonedinto pENTR shuttle vector. The recombinant vector was then iso-lated and used for cloning the 30UTR into destination pANDAvector.

3.3. Recombinant pANDA-3′UTR_B11The 30UTR_B11 insert was cloned from the recombinant pENTR-

30UTR_B11 vector into pANDA vector using LR Clonase enzyme,which is part of the Gateway cloning system (Invitrogen™, USA).Recombination between attR1 sequence from pANDA vector andattL1 sequence from the recombinant pENTR-30UTR_B11 vector, aswell as between attR2 sequence from pANDA vector and attL2sequence from the recombinant pENTR-30UTR_B11 vector causedinsertion of the 30UTR_B11 fragment into pANDA vector. The regionof cloning site in pANDA vector was designed such that the 30UTRinsert can be cloned in two sites with opposite orientation sepa-rated by GUS linker (Figure 4). The recombinant pANDA-30UTR_B11was then introduced into E. coli DH5a.

Introduction of the recombinant pANDA-30UTR_B11 into E. coliDH5a has been successfully carried out. E. coli DH5a transformantcould grow on LA selection media containing 50 ppm kanamycinand 50 ppm hygromycin. Randomly selected colonies were thenverified with colony PCR using primers, HPT-F and HPT-R, devel-oped from hpt gene (Table 1), which is included in the pANDA

M C 1 2 3 4 5 6

500 bp

200 bp

Figure 2. Electrophoregram of colony polymerase chain reaction transformation resultof recombinant pENTR™/D-TOPO® vector with Escherichia coli DH5a bacteria which is199 bp sized 30UTR_B11 fragment (No. 1e6). The fragments were electrophoresed in 1%agarose gel in 1X Tris/borate/EDTA (TBE) buffer at 75 volt for 45 minutes. C ¼ negativecontrol; M ¼ 100 bp marker.

vector. Colony PCR result showed 574 bp sized DNA fragments(Figure 4) suggesting that the B11-RNAi construct has been suc-cessfully introduced into E. coli DH5a. To verify the insert and itsorientation, the recombinant vector was isolated from severaltransformant colonies grown on LB media. Not all positive trans-formants of E. coli DH5a grow on LB media containing both kana-mycin and hygromycin antibiotics. The recombinant pANDA vectorcould only be isolated from two colonies. The recombinant vectorswere then used as template for PCR amplification of GUS linker andthe 30UTR_B11 insert to ensure that the GUS linker and the30UTR_B11 fragment have been inserted in recombinant pANDAvector in proper orientation arrangement. Primers used toobserve the presence of GUS linker were GUS-F and GUS-R primers(collection of Prof. Ko Shimamoto, NAIST, Japan), whereas theprimers used to verify the insert orientation were consisted of fourprimers combinations (Table 2).

The GUS linker verification showed 636 bp DNA fragment,which indicated that the GUS linker has been inserted in therecombinant pANDA vector (Figure 5). However, the GUS linkerposition needs to be further verified whether it resides in propersite in relation to the orientation of the 30UTR_B11 insert. FurtherPCR amplification was done to confirm the 30UTR_B11orientation.

The PCR amplification of the recombinant pANDA-30UTR-B11vector using four primer combinations showed that the 30UTR-B11was inserted in proper orientation separated with GUS linker(Figure 6). The primer combination 1 and 4 (Table 2) identified thetarget insert at the right and left of GUS linker, respectively. Thesizes of the DNA fragment produced from PCR amplification usingboth primer combinations 1 and 4 were ±517 bp (195 bp of3'UTR_B11 þ 322 bp of GUS linker) and ±436 bp (195 bp of3'UTR_B11 þ 241 bp of GUS linker), respectively. The other twoprimer combinations, combinations 2 and 3 (Table 2), did notproduce while within the DNA fragment, because both primercombinations were designed to identify the wrong orientation ofthe target insert. The 30UTR_B11 insert along with the GUS linkerwith proper orientation was then called as B11-RNAi construct.

3.4. Introduction of recombinant pANDA vector intoA. tumefaciens AgL0

The introduction the recombinant pANDA vector intoA. tumefaciens AgL0 has been successfully carried out. Colonies oftransformant A. tumefaciens AgL0 grew on LA selection mediacontaining 50 ppm of kanamycin, 50 ppm of hygromycin, and25 ppm of rifampicin. Colony PCR was performed using the sameprimer combinations performed to identify the orientation oftarget gene in the recombinant pANDA vector (Table 2) and theresult of colony PCR was shown in Figure 7.

Figure 3. Sequence of orientation 30UTR_B11 fragment in pANDA recombinant vector. Yellow color shows the sequence of 30UTR_B11. The grey color shows GUS gene sequence. Thesequence results that the orientation of 30UTR_B11 was inverted repeat.

M 1 2 3 4

500 bp

200 bp

Figure 4. Electrophoregram of colony polymerase chain reaction recombinant pANDAvector using hygromycin primer with fragment size of 574 bp (No. 1e4). The fragmentswere electrophoresed in 1% agarose gel in 1X Tris/borate/EDTA (TBE) buffer at 75 voltfor 45 minutes. M ¼ 100 bp marker.

M 1 4

500 bp636 bp

Figure 5. Electrophoregram of colony polymerase chain reaction recombinant pANDAvector using GUS-F and GUS-R primers (No. 1 and 4). The fragments were electro-phoresed in 1% agarose gel in 1X Tris/borate/EDTA (TBE) buffer at 75 volt for 45 mi-nutes. M ¼ 1 kbp marker

M 1-1 1-2 4-1 4-2 1-3 1-4 4-3 4-4

500 bp

Figure 6. Electrophoregram of polymerase chain reaction result of recombinantpANDA vector using several primer combinations. First number on column numbershows colony number, and second number shows primer combination. The fragmentswere electrophoresed in 1% agarose gel in 1X Tris/borate/EDTA (TBE) buffer at 75 voltfor 45 minutes. M ¼ 100 bp marker.

W. Wahyuningtyas, et al82

Among 12 colonies PCR was tested, four colonies, namely 5, 6, 7,and 8, showed the expected size of the DNA fragment as the frag-ment size from the insert orientation verification. Furthermore, thecolonies were grown in LB liquid selection media containing50 ppm of kanamycin, 50 ppm of hygromycin, and 25 ppm ofrifampicin, then were grown on the LA solid selection with thesame antibiotics. This propagation was to create a sufficientA. tumefaciens AgL0 suspension to infect rice seeds.

3.5. Putative transgenic plant carrying B11-RNAi construct.The B11-RNAi construct has been successfully introduced into

rice cv Hawara Bunar through in-planta infection. Putative trans-genic plants were tested by PCR using GUS-F and GUS-R primersand expected to have 636 bp DNA fragment (Figure 8). Twentyputative transgenic rice were planted on pots in a green house andproduced a total of 112 tillers. Among 20 plants, eight plants wereconfirmed as transgenic plants carrying the B11-RNAi construct.There were no chimera detected in each transgenic plants, whichwas all transgenic plants having all transgenic tillers. If the trans-formation efficiency was calculated based on comparison betweennumber of positive tillers and total tiller evaluated from all trans-formed plants, the transformation efficiency reached 45.53%(Table 3).

4. Discussion

The 30UTR has been successfully isolated from the B11 Al toler-ance gene candidate and cloned into pENTR™/D-TOPO® vector andsubsequently into pANDA vector through the Gateway™ system.Gateway™ technology is an advance cloning technology based onsite-specific recombination reaction of phage lambda. The tech-nology is very easy to construct RNAi vector compared with con-ventional cloning techniques. RNAi technique is an effective way totest the function of the target mRNA in plant biology. Recent de-velopments on the RNAi vector, which uses a constitutive promoter

|---------- 5 ---------| |--------- 6 ----------| M | --------- 7 ----------| |--------- 8 ----------|

Figure 7. Electrophoregram of colony polymerase chain reaction of recombinant pANDA vector using several primer combinations. Number shows colony number, each column isprimer combination. The fragments were electrophoresed in 1% agarose gel in 1X Tris/borate/EDTA (TBE) buffer at 75 volt for 45 minutes. M ¼ 100 bp marker.

M A B C D |------------- 5a-1 ----------| |------------------ 5a-2 ----------------| |----5a-3 ----|

500 bp636 bp

Figure 8. Electrophoregram of polymerase chain reaction transgenic plant using GUS primer A, B, C ¼ negative control (water, IR64, Hawara Bunar WT, respectively). The fragmentswere electrophoresed in 1% agarose gel in 1X Tris/borate/EDTA (TBE) buffer at 75 volt for 45 minutes. D ¼ positive control (pANDA_30UTR_B11). M ¼ 100 bp marker.

Table 3. Transformation efficiency of in-planta Agrobacterium mediated trans-formation technique to rice seeds cv Hawara Bunar

Sample no. Positive tillers Total tillers Efficiency (%)

1b-1 4 4 1001b-2 7 7 1003-2 0 5 03-3 8 8 1003-4 6 6 1003-5 0 2 05a-1 0 8 05a-2 8 8 1005a-3 0 4 05a-4 0 6 05a-5 4 4 1005-4 0 3 05b-1 0 6 05b-2 0 8 05b-3 0 7 05b-4 0 3 06-1 6 6 1006-4 0 6 06-5 0 3 08b-1 8 8 100Total 51 112 45.53

RNAi silencing of aluminum tolerance gene in rice 83

cloning techniques and Gateway™ technologies, makes it easy toconstruct the RNAi vector which has a dsRNA trigger sequences andfacilitates functional analysis of the target gene.

In this new technique, trigger sequence was obtained byamplification using PCR and then be cloned into RNAi vectors withthe desired orientation. One of the RNAi Gateway technologies isthe use of pANDA vector as a destination vector, which has a cornubiquitin1 promoter and GUS linker. The pANDA vector wasdeveloped by Miki & Shimamoto (2004). The vector shows higheffectiveness in disrupting the expression of specific genes andspecific gene family members. The expression of phytoene desa-turase (PDS) gene and GTPase Rac1 gene small family has beensuccessfully suppressed using specific sequences that were clonedinto pANDA vector (Miki et al. 2005). It is expected that B11-RNAiconstruct that used the 30 UTR of B11 gene could silence the B11gene in rice cv Hawara Bunar where the gene was isolated from.

RNAi is RNA that interferes existing RNA function in the cell. Thepresence of RNAi causes RNA in the cell to be bound by RNAi toform a dsRNA. dsRNA will be cut by the enzyme Dicer in thecytoplasm into a short dsRNA fragments size 21e26 nucleotides to

the 30 end of two nucleotide overhangs (Pickford & Cogoni 2003).One of the two strands of each fragment, which is known as theguide strand, was associated with the target mRNA (Hammondet al. 2000). A catalytic component of RNA-induced silencingcomplex called Argonaute (Ahlquist 2002), activates the function ofRNA-induced silencing complex to degrade the target mRNA andsuppress gene expression at various levels (Hannon 2002; Meisterand Tuschl 2004).

The B11-RNAi construct has been introduced to rice cv HawaraBunar through in-planta Agrobacterium mediated transformationtechnique. The Agrobacterium suspensionwas inoculated to the riceseeds. Targets of the in-planta infection in seed are apical meristemcells found in the embryo, or more precisely on the apical meristemof plumula (Lin et al. 2009). However, the embryo in the seedcertainly has shaped organ primordia that have been first differ-entiated. If A. tumefaciens infects the area primordial that arealready differentiated, then most likely generated chimera of T0plants, therefore the T0 generation required strict selection toobtain T1 transgenic seeds. Insertion test should be performed oneach tillers of transformed plants. In this research, selection wasdone by PCR technique using GUS-F and GUS-R primers developedfrom GUS linker sequence. Based on the insertion test, it could befound that one plant has transgenic and non-transgenic tillers(chimera), but there is also possibility that the whole tillers aretransgenic. Careful infection technique greatly affects the success ofthe infection and can be one of the factors affecting variation oftransformation efficiency among genotypes. In addition, differentresponses to the infection among plants or genotypes alsocontribute to the transformation efficiency. The infection result willbe the best if target is on plumula meristem. If plumula is pierceddirectly (not plumula meristem), there will be a fatal error and seedwill fail to germinate (Lin et al. 2009). This research has obtainedeight positive transgenic plants with all transgenic tillers indicatedno chimera produced. The transformation efficiency was 45.53%(Table 3).

The transgenic rice plants will then be grown to produce T1seeds, which subsequently will be used for further Al toleranceanalysis. It is expected that the transgenic plant carrying B11-RNAiconstruct will be susceptible to Al stress than that of wild-type cvHawara Bunar that is originally tolerant to Al stress. If this is truethen the B11 gene is confirmed to have a role in Al tolerancemechanism in rice. It could be one of the Al tolerance genes in ricethat could be used to develop Al tolerant rice cultivar.

W. Wahyuningtyas, et al84

Conflict of interest

There is no conflict of interest.

Acknowledgements

The research was funded by Higher Education Research Excel-lence Grant, scheme Basic Research for Division, Bogor AgriculturalUniversity, The Indonesian Ministry of Research, Technology, andHigher Education, F.Y. 2013, contract number 264/IT3.41.2/L2/SPK/2013 with PIC: Dr. Miftahudin.

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